Hematopoietic in vitro assays
Our most popular service involves the hematopoeitic colony-forming cell (CFC) assay, an assay that has been validated for the determination of maximum tolerated doses (MTD) by ECVAM. 1,2
We use STEMCELL’s MethoCult ®
product line, recognized as the global gold standard for the detection and quantification of hematopoietic progenitors using CFC assays.
Hematopoietic CFC assays can be used to examine simultaneously the effects of substances (lead compounds/clinical candidates, mimetics, cytokines or chemokines) on the processes of proliferation and differentiation. Hematopoietic progenitors from different species can be assessed:
Evaluation of Stimulation
Substances thought to stimulate and/or enhance proliferation of hematopoietic progenitors can be tested for functionality in the CFC assay. FIGURE 1: Stimulation by Erythropoietin (EPO)
Stimulation of erythroid progenitor proliferation by erythropoietin (EPO) is both quantifiable (Figure A) and qualitative (Figure B, colony size increases with increasing [EPO]). In vivo
models can be used to confirm stimulatory activity of molecules.
Evaluation of Inhibition or Toxicity
Substances thought to inhibit the proliferation and/or differentiation of hematopoietic progenitors can be screened to assess their effects on the different lineages of progenitors: myeloid, erythroid, and/or megakaryocytes using the CFC assay. Both IC 50
and IC 90
values can be generated to rank test compounds. TABLE: Comparison of myelotoxicity (CFU-GM) in different species FIGURE 2: Determination of IC50 values for 5-Fluorouracil
Dose response curves and IC50
values for both human BM derived erythroid and myeloid progenitors incubated with 5-Fluorouracil FIGURE 3: Human BM Colonies in the presence of a toxic compound
The size of CFCs will change in the presence of an inhibitory compound, serving as a sensitive toxicity assay readout.
Neural in vitro assays
STEMCELL scientists have worked with leaders in the neural stem cell field to develop standardized reagents and assays for the culture of neural stem and progenitor cells.
are highly dynamic, three-dimensional floating spheres containing cells from different stages of neural development – neural stem cells, progenitors and mature cells. These neurospheres are the basis of both our proliferation (NCFC) assay and differentiation assay.
We use the NCFC assay to assess the effects of your substance of interest on primary neural cells. FIGURE 1: Assays for neural stem and progenitor cells
Mesenchymal in vitro assays
Drug candidates destined for the tissue engineering market can be assessed for stimulatory or inhibitory effects on the bone marrow microenvironment (mesenchymal cell matrix). These cells, under the appropriate conditions, can differentiate into structural cell types that make up adipose, cartilage, bone and muscle.
Functional assays using mesenchymal stem and progenitor cells from human bone marrow or mouse compact bone can be used to quantify and qualitatively assess the effects of compounds:
- Measure the effects of test compound on progenitor frequency
- Assess progenitor proliferative potential or overall expansion potential
- Quantitation of mesenchymal progenitor content in bone marrow
- Test the differentiation potential of mesenchymal stem cells into adipose, osteogenic and chrondrocyte lineages
The Contract Assay Services group has developed new assays for the quantification of mouse CFU-F from compact bone, a source known to be richer than the bone marrow in mesenchymal stem and progenitor cells. Mouse CFU-F frequency increases when culturing compact bone-derived MSCs under low oxygen conditions.
FIGURE 1: Effects of Doxorubicin on Human CFU-F
Dose response curve and IC50 value for human BM derived CFU-F incubated with Doxorubicin. Inhibitory compounds can affect the size and density of the CFU-F colony.